Project Summary Flaviviruses are insect-transmitted human pathogens that most notably cause Zika-associated microcephaly, dengue fever and West Nile fever. This study aims to elucidate the molecular mechanisms of the flaviviral nonstructural protein 1 (NS1), a multi-functional virulence protein. Intracellular NS1 is essential to replication of the viral RNA genome, whereas secreted NS1 interacts with innate immunity proteins and, in some cases, induces disease phenotypes. Our crystal structures of full-length, glycosylated NS1 from Zika virus (ZIKV), dengue virus serotype 2 (DENV2) and West Nile virus (WNV) will guide experiments to identify which of the distinct domains of NS1 are responsible for which of its several functions. Despite their overall similarity (~50% identical amino acid sequences), several properties specific to individual flaviviruses are attributed to the NS1 proteins, making comparative analysis especially powerful in dissecting the molecular functions of NS1. An extensive panel of mutants based on comparative mutagenesis of DENV2 and WNV NS1 will be expanded to include ZIKV NS1. Based on observations in the initial comparative study that NS1 affects virus particle assembly, we will test the hypothesis that NS1 acts as an infectivity factor by aiding viral structural protein folding or virus particle transit through the secretory system. Intracellular NS1 is localized to the ER lumen as a membrane-associated dimer with a critical role in replication through association with viral transmembrane proteins. We will probe the interaction with viral protein NS4B through mutagenesis and biophysical experiments. Electron cryo-microscopy or crystallography will be used to investigate the structure of secreted NS1, a hexameric lipo-protein particle. An initial observation that NS1 remodels membranes will be followed by detailed experiments using light microscopy with fluorescently tagged lipids to determine any lipid preference in this key association. Binding experiments will identify which domains of NS1 interact with which domains of two proteins of the complement system and the innate immunity Toll-like receptor 4. The results will provide a foundation for development of antiviral drugs and/or effective vaccines, which are not available or of limited use for Zika, dengue or West Nile viruses.